Today, many solid pediatric cancers have a poor prognosis, contributing to an immense amount of physical and emotional suffering on the parts of the victims and their families. A key development on the pathway to addressing this unmet medical need has been growing recognition that the cellular bases of most pediatric cancers are different from those regulating their adult counterparts. Therefore, drug discovery efforts focused on the identification of new therapeutic leads that target the mechanisms underpinning pediatric cancers are needed. Fundamental to the success of any effort for improving chemotherapeutic options against pediatric cancers is an approach that matches the discovery possibilities embodied in a chemically diverse library of drug-like molecules with assays probing the selectivity/specificity of disease-relevant targets. Natural products fulfill this key criterio offering a rich assortment of novel structural features that nature has evolved into bioactive compounds. Our preliminary studies demonstrate that new fungal taxa derived from Great Lakes sediments generate compounds with these types of attractive biological profiles as exemplified in preliminary studies demonstrating selective activity against pediatric cancer cell lines. Based on these data, we hypothesize that the Great Lakes contain a unique and diverse assemblage of fungi that is virtually untapped for the production of bioactive natural products with potential against childhood cancers. To achieve the goal of transforming this fungal source of biological and chemical diversity into therapeutically-promising molecules, we have assembled a collaborative network of researchers with expertise in natural products chemistry, cancer drug discovery/pharmacology, Great Lakes ecology, and mycology. We will test the central hypothesis and fulfill the objectives of this proposal through six specific aims: 1) procuring sediment samples from diverse Great Lakes environments, 2) isolating fungi from sediment samples and preparing an extract library for screening, 3) testing extracts for activities in a suite of new and disease-relevant-assays pertaining to rare yet fatal pediatric cancers, 4) prioritizing bioactive samples for scale-up studies using bioassay data, taxonomic affinity, and chemical markers, 5) purifying bioactive compounds, and 6) verifying the in vivo activities of purified compounds in xenograft mouse models. Several innovative features have been built into our approach that pertain to both the biological assays (focus on selective inhibition of enzymatic and cell targets exhibited by pediatric cancers) and natural products chemistry approaches (new methods for the semi-automated isolation of fungi, expedited extract preparation, and advanced dereplication tools). These new techniques are expected to enhance our approach to identifying new bioactive compounds. Our research is significant because many pediatric cancers remain untreatable and vigorous efforts are needed to identifying compounds with promising therapeutic profiles that address this unmet medical need. We expect that that our studies will provide several new options for further development as therapies for childhood cancers.